ABSTRACT The long-term aim of this research is to develop an efficient treatment against severe malaria. Specific adhesion of P. falciparum parasite-infected erythrocytes (IE) in deep vascular beds can result in severe complications, such as cerebral malaria, placental malaria, respiratory distress, and severe anemia. Even timely treatment with currently available parasite-killing drugs fails in 10?15% of severe malaria cases. Treatment with adjunct drugs that reverse and prevent parasite adhesion may significantly improve the outcome and save lives. The main idea of using anti-adhesion drugs in severe malaria is to release sequestered parasites (or prevent additional sequestration) as quickly as possible. This is extremely critical for survival because it would decrease local and systemic inflammation associated with severe disease, and help to reestablish the microvascular blood flow providing immediate relief to the patient. Concomitantly, patients would be treated with common drugs that kill parasites. This proposal is to identify individual compounds with low (<100 nM range) IC50 which inhibit cytoadhesion of IE to the host placental and brain microvasculature receptors and are suitable for development into effective adjunct drugs to treat placental and cerebral malaria, which is supported by strong preliminary data. This multi-PI proposal is based on the success of our recently developed high throughput screening (HTS) approach for identification of anti-adhesion compounds (supported by previous R56 funding) and large combinatorial libraries (~30x106 compounds) developed at Torrey Pines Institute for Molecular Studies (TPIMS). Our two-step HTS approach is specifically designed to work with compound mixtures to make screening and deconvolution to single active compounds fast and efficient. Specific aims of the project are: 1) De-convolute most active mixtures to identify individual compounds, and validate their anti-adhesion activity for inhibiting interactions implicated in placental and cerebral malaria using live IE. This will be done using well-validated target receptors CSA (placental malaria) and ICAM-1 (cerebral malaria), and corresponding receptor-binding domains of a family of parasite proteins PfEMP1, expressed and characterized in our previous work. Deconvolution to single active compounds (hits) is performed by 2 or 3 iterative screens on bead-bound domains of positional libraries with 100 ? 1000 mixtures/compounds. These single hits will then be synthesized, purified, characterized, and validated for IE binding inhibitory activity and cytotoxicity with live IE (lab and field) and mammalian cells. 2) Demonstrate therapeutic potential of identified anti-adhesion compounds in vitro. We will measure a number of inflammation markers, produced/changed in response to cytoadhesion of IE to vascular cells, including markers identified previously for HUVEC (with ICAM1 binding IE) and BeWo (with CSA binding IE). Returning the inflammation markers toward normal levels after treatment of IE/(BeWo or HUVEC) with anti-adhesion hits will validate their therapeutic potential in vitro. Successful completion of these Aims will provide an avenue for future hit to lead discovery of anti-adhesion drugs for adjunct treatment of severe malaria.